Helicobacter pylori colonizes the normal acid-secreting stomach of about 50% of the world's population. Colonization is associated with gastric disease, including gastritis, peptic and duodenal ulcers, gastric carcinoma, and MALT lymphoma. Although initial eradication rates with triple therapy were successful (~95%), antibiotic resistance has made successful treatment of infection progressively more difficult. Thus, there is a critical need for an antibiotic-free alternative eradication mono-therapy that is . pylori-specific, sparing commensal gut flora or improvement to antibiotic therapy that decreases the duration and complexity of treatment. The long-term goal of this study is to understand how H. pylori responds to and survives gastric acidity and exploit this knowledge to develop novel treatment regimens and/or improve current eradication therapies. The objective here is to determine how the acid-sensitive Two-Component System (TCS), ArsRS, regulates the acid-induced trafficking of urease and its accessory proteins to the inner membrane to form a membrane- bound complex with UreI that is required for gastric infection and acid survival. The rationale for the study is that understanding the mechanisms used by ArsS will allow pharmacological interference resulting in new H. pylori-specific antibiotic-free monotherapy and/or improvement in current eradication regimens. The specific aims are: 1. Elucidate the acid-induced signaling mechanism mediated by the sensor kinase ArsS. Because ArsS is essential for gastric colonization and acid survival, knowing the mechanism of protein trafficking to UreI and the formation of the UreI/urease membrane complex will provide novel eradication targets. 2. Identify the acid-dependent membrane proteome and the contribution of ArsS to intrabacterial trafficking of the identified proteins. This remarkable example of intra-bacterial protein trafficking provides a unique opportunity to develop anti-infective drugs. 3. Identify high affinity ArsS inhibitors by High- Throughput Screening (HTS). Because the acid-sensing domain of ArsS is in the periplasm and ArsS is essential for gastric infection, it is an attractive eradication target. We have designed an HTS that will allow detection of inhibitors that select between ArsS and UreI. The work of aims 1 and 2 will use biochemical and molecular biological techniques to identify proteins and regulatory pathways responsible for maintenance of gastric colonization by H. pylori. The work proposed in aim 3 will provide lead compounds that inactivate the ArsRS signaling cascade required for gastric infection. The results of these three aims will have an important positive impact by providing novel targets and improving current therapies for eradication.